In eukaryotes, the regulated progression through the cell cycle is controlled by a group of genes whose expression fluctuates throughout the cycle. Cyclin dependent kinases (CDKs) and their associated regulatory subunits, the cyclins, are the primary regulators of the cell cycle. These heterodimeric complexes act by phosphorylating downstream targets that, in turn, trigger signaling events that liberate nuclear proteins necessary for entry into subsequent phases of the cell cycle. Contributing to the activation and inactivation of these complexes is a family of protein tyrosine kinases known as polo-like kinases or Plks reviewed in (Nigg, Curr. Opin. Cell. Biol., 1998, 10, 776-783).
As cells progress through the cell cycle, polo-like kinases undergo fluctuations in abundance, activity and localization thereby controlling multiple stages of the cell cycle (Hamanaka et al., J. Biol. Chem., 1995, 270, 21086-21091). This family of kinases also function in centrosome maturation (Lane and Nigg, J. Cell. Biol., 1996, 135, 1701-1713), bipolar spindle formation (Golsteyn et al., J. Cell. Biol., 1995, 129, 1617-1628), DNA damage checkpoint adaptation (Arnaud et al., Chromosoma, 1998, 107, 424-429) and regulation of the anaphase-promoting complex (Kotani et al., Mol. Cell, 1998, 1, 371-380)
It is currently believed that a number of diseases and/or disorders are a result of either aberrant expression or functional mutations in the molecular components of kinase cascades. Consequently, considerable attention has been devoted to the characterization of these proteins.
Polo-like kinase (also known as polo-like kinase 1, plk-1, plk and stpk13) was the first member of this family to be identified as the mammalian counterpart to the Drosophila melanogaster gene, polo, required for passage through mitosis (Golsteyn et al., J. Cell. Sci., 1994, 107, 1509-1517; Hamanaka et al., Cell. Growth Differ., 1994, 5, 249-257; Holtrich et al., Proc. Natl. Acad. Sci. U.S. A., 1994, 91, 1736-1740; Lake and Jelinek, Mol. Cell. Biol., 1993, 13, 7793-7801). Expression of polo-like kinase was shown to correlate with mitotic activity of cells (Golsteyn et al., J. Cell. Sci., 1994, 107, 1509-1517; Lake and Jelinek, Mol. Cell. Biol., 1993, 13, 7793-7801) and to be high in tumors of several origins including lung, colon, stomach, smooth muscle, and esophagus (Holtrich et al., Proc. Natl. Acad. Sci. U.S. A., 1994, 91, 1736-1740). Overexpression or constitutive expression of polo-like kinase has also been shown to induce malignant transformation of mammalian cells (Mundt et al., Biochem. Biophys. Res. Commun., 1997, 239, 377-385; Smith et al., Biochem. Biophys. Res. Commun., 1997, 234, 397-405). Microinjection of polo-like kinase antisense RNA into growing mouse NIH3T3 cells was shown to block tritiated thymidine incorporation, suggesting that polo-like kinase expression is restricted to and required by proliferating cells (Hamanaka et al., Cell. Growth Differ., 1994, 5, 249-257).
Further support of this conclusion is found in studies showing that elevated levels of polo-like kinase expression are significant prognostic indicators of non-small cell lung cancer (Wolf et al., Oncogene, 1997, 14, 543-549), breast and lung cancer (Yuan et al., Am. J. Pathol., 1997, 150, 1165-1172), esophageal carcinoma (Tokumitsu et al., Int. J. Oncol., 1999, 15, 687-692) and squamous cell carcinomas of the head and neck (Knecht et al., Cancer Res., 1999, 59, 2794-2797).
The pharmacological modulation of polo-like kinase activity, expression, or function may therefore be an appropriate point of therapeutic intervention in pathological conditions.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of polo-like kinase and investigative strategies aimed at modulating polo-like kinase function have involved the use of antibodies, and antisense expression vectors and antisense oligonucleotides. Elez et al. used an antisense strategy to inhibit cell proliferation of cancer cells in cell culture and in the nude mouse. In these studies one phosphorothioate antisense oligonucleotide inhibited the expression of polo-like kinase in culutured A549 cells leading to the loss of cell viability and anti-tumor activity in nude mice A549 xenografts (Elez et al., Biochemical and Biophysical Research Communications, 2000, 209, 352-356).
However, these strategies are untested as therapeutic protocols and consequently there remains a long felt need for agents capable of effectively inhibiting polo-like kinase function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of polo-like kinase expression.
The present invention provides compositions and methods for modulating polo-like kinase expression.